Myelodysplasic syndromes (MDS) are clonal disorders of ineffective hematopoiesis that commonly progress to acute leukemia. The primary mechanisms underlying aberrant hematopoietic differentiation have not been identified. Therapeutic options for patients with MDS are limited, and the development of novel therapies has been inhibited by the paucity of validated therapeutic targets and the absence of cell culture or animal models of the disease. A major barrier to the elucidation of the molecular pathophysiology of MDS has been the absence of a systematic experimental approach to identify genes within the large cytogenetic deletions commonly associated with MDS. The development of RNA interference in mammalian cells offers a novel strategy for investigating the function of genes. We have designed and validated a functional screen using short interfering RNAs (siRNAs) to decrease the expression of each gene within the 5q deletion, a common cytogenetic abnormality in MDS associated with a defined clinical phenotype. Patients with MDS and a 5q deletion as the sole cytogenetic abnormality have a severe refractory anemia. We are therefore testing the effect of each siRNA on erythroid differentiation of human CD34+ cells in vitro. Candidate genes will be validated using a variety of in vitro functional assays of differentiation and proliferation. In addition, candidate genes will be validated in vivo by performing bone marrow transplants in mice using donor cells that express siRNAs targeting candidate genes. We plan to complement these functional studies with a detailed genetic analysis of candidate genes in patient samples using microarry-based comparative genome hybridization and high throughput sequencing. Using this combination of genomic approaches, we hope to identify the critical gene or genes causing abnormal differentiation in the 5q- syndrome. This finding would provide a critical foothold towards a greater understanding of myelodysplastic syndromes, the multi-step pathogenesis of hematologic malignancies, and disorders of erythroid differentiation.